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PubMedDate not supplied

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum.

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.

Animals↗
PubMed1997

[Sialic acid determinations of serum and urine specimens in calcium-containing urinary stone formers].

PURPOSE: The aim of the present study was to compare sialic acid concentrations of serum and urine specimens in both calcium (Ca)-containing urinary stone formers and non-stone formers. Moreover, we studied inhibitory activity of sialic acid upon the calcium oxalate (CaOx) crystal aggregation and growth. MATERIALS AND METHODS: Sialic acid determinations were done on fresh serum and urine samples of 35 Ca-containing urinary stone formers (stone formers group) and 20 non-stone formers (patient controls group). Inhibitory activity of sialic acid upon the CaOx crystal aggregation and growth was studied by using in vitro assay method of seed crystal system. RESULTS: Serum sialic acid concentrations were found to be similar in the two groups. Urinary sialic acid concentrations were significantly lower in the urine specimens of stone formers than in their patient controls. Sialic acid showed a dose dependent inhibitory activity upon the CaOx crystal aggregation and growth into seed crystal method. CONCLUSION: It is suggested that urinary sialic acid may play some role during the phase of stone formation from the results of the present study, because sialic acid shows marked inhibitory activity upon the CaOx crystal aggregation and growth at concentrations higher than 100 mg/dl.

Aged↗
PubMed1997

Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-beta-D-glucopyranoside and related compounds.

Resveratrol 3-O-beta-D-glucopyranoside (1) has been isolated from the seeds of Erythrophleum lasianthum (Caesalpinioidae, Leguminosae), a South African plant used in traditional medicine, and has shown antiplatelet aggregation activity. The synthesis of 1, related hydroxystilbenes, and their glucosides has been undertaken to provide larger quantities, for further biological evaluation, and has been accomplished via Wittig reactions followed by glucosylation under phase transfer catalysis.

Glucosides↗
PubMed1994

Light-scattering investigations of nucleation processes and kinetics of crystallization in macromolecular systems.

Quasi-elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, sigma = ln(c/s) where c and s are concentration and solubility of protein, varied from approximately 0.1 for pumpkin seed globulin to approximately 0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation-dependent critical nuclear size, R(c), for different macromolecular systems was estimated from time-dependent size-distribution analyses to be in the range of approximately 10(3) for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interfacial free energy was deduced to be 3.3-9.2 kJ mol(-1). These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3-50 kJ mol(-1). Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubility versus solution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early stages of the aggregation process.

Journal Article↗
PubMed2005

The decellularized porcine heart valve matrix in tissue engineering: platelet adhesion and activation.

An approach in tissue engineering of heart valves is the use of decellularized xenogeneic matrices to avoid immune response after implantation. The decellularization process must preserve the structural components of the extracellular matrix to provide a biomechanically stable scaffold. However, it is known that in vascular lesions platelet adhesion to extracellular matrix components occurs and platelet activation is induced. In the present study we examined the effects of a decellularized porcine heart valve matrix on thrombocyte activation and the influence of re-endothelialisation in vitro. Porcine pulmonary conduits were decellularized using Triton X-100, Na-deoxycholate and Igepal CA-630 followed by a ribonuclease digestion. Cryostat sections of decellularized heart valves with and without seeding with human umbilical vein endothelial cells (HUVEC) were incubated with platelet rich plasma. Samples were either stained with fluorescent antibodies for CD41 and PAC-I (recognizing the activated fibrinogen receptor) or fixed with glutaraldehyde. Thereafter, the samples were processed for laser scanning microscopy (LSM) or scanning electron microscopy (SEM). Examination by LSM showed numerous platelets with co-localized staining for CD41 and PAC-1 on the nonseeded decellularized heart valve matrix whereas after seeding with endothelial cells no platelet activation was detected. SEM revealed platelet adhesion and aggregate formation only on the surface of the non-seeded or partially denuded matrix specimens. We show in this study that the decellularized porcine matrix acts as a platelet-activating surface. Seeding with endothelial cells effectively abolishes the platelet adhesion and activation and therefore is necessary to eliminate thrombogenicity in tissue engineered heart valves.

Animals↗
PubMed2002

Comparative study of Venostasin and Pycnogenol in chronic venous insufficiency.

The aim of this study was to compare the efficacy of Venostasin (horse chestnut seed extract) and Pycnogenol (French maritime pine bark extract) in the treatment of chronic venous insufficiency (CVI). In an open, controlled comparative study 40 patients with diagnosed CVI were treated either with 600 mg chestnut seed extract per day or 360 mg Pycnogenol per day over a period of 4 weeks. The following parameters were investigated before the start of treatment and after 2 and 4 weeks of treatment: circumference of the lower legs and rating of subjective symptoms (scores) of pain, cramps, night-time swelling, feeling of "heaviness", and reddening of the skin. In addition, blood levels of cholesterol LDL and HDL were determined before and at the end of treatment. Pycnogenol significantly reduced the circumference of the lower limbs and significantly improved subjective symptoms. Furthermore, Pycnogenol significantly decreased cholesterol and LDL values in the blood, whereas HDL remained unaffected. Venostasin only moderately but not significantly, reduced the circumference of the lower limbs and marginally improved symptoms. Venostasin had no influence on the determined lipid values. Both medications were equally well tolerated. In conclusion, Pycnogenol was found to be more efficacious than Venostasin for the treatment of CVI.

Adult↗
PubMed2001

Mechanism of prion loss after Hsp104 inactivation in yeast.

In vivo propagation of [PSI(+)], an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104. Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation. Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele. In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of [PSI(+)] and another candidate yeast prion, [PIN(+)]. Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation. The [PSI(+)] response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased. A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35(PSI+) aggregates and an increase in their size. This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle. Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).

Adenosine Triphosphatases↗
PubMed1990

Localization of two fibronectin-binding glycoproteins in rat liver and primary hepatocytes. Co-distribution in vitro of integrin (alpha 5 beta 1) and non-integrin (AGp110) receptors in cell-substratum adhesion sites.

We have compared the localization of integrin alpha 5 beta 1 and AGp110 (apical glycoprotein of Mr 110 x 10(3]] in rat liver parenchyma and in primary hepatocyte cultures. Integrin alpha 5 beta 1 is a heterodimeric fibronectin receptor. AGp110 is a newly described monomeric glycoprotein of the apical (bile canalicular) membrane domain of liver parenchyma that binds in an RGD-independent manner to fibronectin and mediates spreading of hepatocytes onto fibronectin-coated substrata. Using Western blotting of fractionated liver membranes and immunocytochemistry of liver sections at light- and electron-microscope levels, we have confirmed that AGp110 is a canalicular glycoprotein and have established that integrin is located in approximately equal proportions in the sinusoidal, lateral and canalicular membrane domains. In the canalicular surface domain both glycoproteins are associated with microvilli. Examination of immunolabelled primary hepatocytes spread on fibronectin-coated substrata by light and laser scanning confocal microscopy revealed colocalization of AGp110, integrin, actin and vinculin in substratum-attached microextensions at the periphery of the basal cell surface. Actin filaments that terminated at these cell processes originated from circular sub-cortical actin fibres. Interference reflection microscopy revealed focal adhesive contacts at the edge of the basal cell periphery at the same location where AGp110 and integrin were observed by immunofluorescence. In vitro, a proportion of the primary hepatocytes seeded onto fibronectin-coated substrata aggregated into colonies of several cells with intercellular contacts between neighbouring cells. Cell-substratum contacts containing integrin, AGp110, actin and vinculin followed the contours of these colonies in the same manner as they delineated the basal periphery of single, substratum-attached cells. We conclude that both integrin and AGp110 contribute to hepatocyte-fibronectin adhesive interactions and that intercellular adhesion and cooperation among hepatocytes in their response to fibronectin matrices leads to colony formation and morphological differentiation of parenchymal cell monolayers in vitro.

Actins↗
PubMed2002

Alzheimer beta-amyloid peptides: normal and abnormal localization.

Alzheimer's disease (AD) neuropathology is characterized by accumulation of "senile" plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are principally composed of aggregates of up to 42/43 amino acid beta-amyloid (A beta) peptides. The discovery of familial AD (FAD) mutations in the genes for the amyloid precursor protein (APP) and presenilins (PSs), all of which increase A beta42 production, support the view that A beta is centrally involved in the pathogenesis of AD. A beta42 aggregates readily, and is thought to seed the formation of fibrils, which then act as templates for plaque formation. A beta is generated by the sequential intracellular cleavage of APP by beta-secretase to generate the N-terminal end of A beta, and intramembranous cleavage by gamma-secretase to generate the C-terminal end. Cell biological studies have demonstrated that A beta is generated in the ER, Golgi, and endosomal/lysosomal system. A central question involving the role of A beta in AD concerns how A beta causes disease and whether it is extracellular A beta deposition and/or intracellular A beta accumulation that initiates the disease process. The most prevalent view is that SPs are composed of extracellular deposits of secreted A beta and that A beta causes toxicity to surrounding neurons as extracellular SP. The recent emphasis on the intracellular biology of APP and A beta has led some investigators to consider the possibility that intraneuronal A beta may directly cause toxicity. In this review we will outline current knowledge of the localization of both intracellular and extracellular A beta.

Alzheimer Disease↗
PubMedDate not supplied

Propagation of human nasal chondrocytes in microcarrier spinner culture.

OBJECTIVE: The aim of this study was to test the effectiveness of nasal septal chondrocytes, propagated in microcarrier spinner culture, as an alternative tissue source of chondrocytic cells for cartilage grafts for head and neck surgery and for articular cartilage repair. METHODS: We harvested chondrocytes from 159 patients, ranging in age from 15 to 80 years and undergoing repair of a deviated nasal septum, and propagated the cells in a microcarrier spinner culture system. The nasal chondrocytes proliferated and produced extracellular matrix components similar to that produced by articular chondrocytes. RESULTS: In microcarrier spinner culture on collagen beads, chondrocyte numbers increased up to 14-fold in 2 weeks. After a month, the microcarriers seeded with nasal chondrocytes began to aggregate, producing a dense cartilage-like material. The newly synthesized extracellular matrix was rich in high molecular weight proteoglycans, and the chondrocytes expressed type II collagen and aggrecan but not type I collagen. CONCLUSION: These studies support the feasibility of engineering cartilage tissue using chondrocytes harvested from the nasal septum. Injectable and solid formulations based on this technology are being evaluated for applications in craniomaxillofacial reconstructive surgery and for plastic and orthopedic surgery practices.

Adolescent↗
PubMed2001

Formation of human fibroblast aggregates (spheroids) by rotational culture.

In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast-material interactions, and augmenting cell-cell interactions. In addition, to promote cell-cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 microm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24-36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid. which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.

Cell Aggregation↗
PubMed2006

Seed dispersal and spatial pattern in tropical trees.

Theories of tropical tree diversity emphasize dispersal limitation as a potential mechanism for separating species in space and reducing competitive exclusion. We compared the dispersal morphologies, fruit sizes, and spatial distributions of 561 tree species within a fully mapped, 50-hectare plot of primary tropical forest in peninsular Malaysia. We demonstrate here that the extent and scale of conspecific spatial aggregation is correlated with the mode of seed dispersal. This relationship holds for saplings as well as for mature trees. Phylogenetically independent contrasts confirm that the relationship between dispersal and spatial pattern is significant even after controlling for common ancestry among species. We found the same qualitative results for a 50-hectare tropical forest plot in Panama. Our results provide broad empirical evidence for the importance of dispersal mode in establishing the long-term community structure of tropical forests.

Animals↗
PubMed1999

Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture.

Deposition of aggregated amyloid beta-protein (Abeta), a proteolytic cleavage product of the amyloid precursor protein (Abeta ), is a critical step in the development of Alzheimer's disease(Abeta++). However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo. Here, we report that a seeding Abeta, which catalyzes the fibrillogenesis of soluble Abeta, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.

Amyloid beta-Peptides↗
PubMedDate not supplied

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices.

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.

Biocompatible Materials↗
PubMed2003

Role of alpha-synuclein carboxy-terminus on fibril formation in vitro.

Alpha-synuclein (alpha-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble alpha-syn into filamentous aggregates may contribute to disease pathogenesis. Since mechanisms leading to the formation of alpha-syn inclusions are unclear, in vitro models of alpha-syn aggregation may yield insights into this process. To that end, we examined the consequences on the progressive deletion of the carboxy-terminus of alpha-syn in regulating fibril formation, and we show here that carboxy-terminal truncated alpha-syn proteins aggregate faster than the full-length molecule. Protease digestion and immunoelectron microscopy indicate that the alpha-syn amino- and carboxy-termini are more solvent exposed than the central core and that filaments formed from carboxy-terminal truncated alpha-syn are narrower in diameter than the full-length molecule. Moreover, seeding experiments under conditions where full-length alpha-syn did not readily aggregate revealed that carboxy-truncated alpha-syn extending from amino acids 1-102 and 1-110 but not 1-120 were efficient in seeding full-length alpha-syn aggregation over a range of concentrations. Using site-directed mutagenesis, the negatively charged residues 104, 105 and 114, 115 in the carboxy-terminus were implicated in this reduced aggregation and the lack of seeding of full-length alpha-syn fibrillogenesis by 1-120. Our data support the view that the middle region of alpha-syn forms the core of alpha-syn filaments and that negative charges in the carboxy-terminus counteract alpha-syn aggregation. Thus, the carboxy-terminus of alpha-syn may regulate aggregation of full-length alpha-syn and determine the diameter of alpha-syn filaments.

Actin Cytoskeleton↗
PubMed2003

Growth of endophyte, Neotyphodium, and its host plant, tall fescue (Festuca arundinacea), under 3D-clinorotation.

Growth of a filamentous fungus endophyte, Neotyphodium, and its host plant, tall fescue, Festuca arundinacea, was examined during the seed germination process under pseudo-microgravity [correction of micrgravity] generated by three dimensional (3D-) clinorotation. The shoot growth of tall fescue infected with the endophyte was remarkably suppressed on a 3D-clinostat compared with that of the ground control. Without being infected, shoot growth of tall fescue was not strongly affected by the 3D-clinorotation. Many aggregated hyphae were observed in the plant seed incubated for 1-day on the 3D-clinostat [correction of clinost] than in those kept on the ground. These results indicate that the clinorotation induces responses in the endophyte and its host plant different from those under normal gravity.

Festuca↗
PubMed1999

Progressive maturation resistance to microcystin-LR cytotoxicity in two different hepatospheroidal models.

To develop three-dimensional (3D) cytotoxicity models further, microcystin-induced cytoskeletal disruption was tested in two different models of multicellular hepatocyte aggregate formation (hepatospheroids). Rat hepatocyte suspensions were seeded either onto poly(2-hydroxyethylmethacrylate)-treated culture wells (poly-HEMA) or in a rotating wall vessel (RWV) device which provides minimal shear forces and enhances differentiated 3D growth. Ninety percent of spheroids forming on poly-HEMA tended to fuse and form nonhomogeneous multilobular structures by day 4 of incubation. In contrast, spheroids cultured in the low-shear environment formed homogeneous aggregates that averaged 126 + 10 microm diameter in size at day 7. Microcystin-LR (10(-6) mol/L) was put into contact (90 min in serum-free medium) with hepatocyte suspensions and spheroids formed in both systems for 1, 4 or 7 days. As already described, microcystin-LR (after 90 min), induced cytoskeletal disruptions (blebs) in 98% of the isolated primary hepatocytes maintained in suspension. In 3D cultures, blebs were detected only on poly-HEMA nonhomogeneous early prespheroids. All other mature spheroids (poly-HEMA or RWV) exposed to the toxin did not exhibit obvious morphological signs of toxicity. Moreover, microcystin-LR pre-incubation with hepatocyte suspension prevented the formation of conventional spheroids. In conclusion, the low-shear, simulated-microgravity environment generated high yields of regularly engineered spheroids. In both models, progressive resistance of mature spheroids to microcystin-LR-induced cell deformation developed with time in culture. Microcystin-LR inhibition of the formation of rat hepatospheroids in isolated hepatocyte suspension could be used as a complementary biological assay for detection of the presence of biologically active microcystin-LR in water samples.

Animals↗